Plane-wave focusing, resolution limit

It can be derived from the transport of intensity equation that this signal has two terms at the edges of the pupil it is proportional to the local wavefront gradient in the direction normal to the pupil edge (i.e. radial in the case of a circular pupil) and elsewhere in the pupil it is proportional to the local wave-front curvature (Roddier, 1988). The signal is more intense when the planes are nearer the telescope focus, but diffraction will limit the spatial resolution more. Thus there is a trade-off between resolution and signal-to-noise (see Ch. 24). 

The major advantage of TIRF is that fluorophores outside the evanescent wave (typically more than 200 nm away from the surface) are not excited. Hence, TIRF has an intrinsic sectioning capability. Of interest is that the section capability (z-resolution) is far better than for confocal microscopy systems, which typically have a z-resolution of about 1 /mi. In addition and in contrast to confocal microscopy, TIRF does not cause out-of-focus bleaching because only the molecules at the surface will sense the evanescent wave. However, in comparison with confocal microscopy, a clear limitation of TIRF is that only one z-plane can be imaged the molecules immediately adjacent to the surface. As a consequence,... 

In the optical microscope the Zernike phase contrast method is applied to convert the phase modulation into a contrast, by adding a phase plate in the back focal plane. This is very close to the wave aberration at the Scherzer focus, but the performance of the electron microscope is rather limited. Even the minimum Cs available today does not allow a carbon atom to be visualized. The solution is to add a new spherical corrected objective lens for sub-angstrom resolution. Figure 13 shows the optical path of a beam in a TEM working under holographic conditions. 

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